Molecular UV-Visible Spectroscopy
Lecture Date: January 30th, 2013
Electronic Spectroscopy (Review)
 Spectroscopy of the electrons surrounding an atom or a
molecule: electron energy-level transitions
Atoms: electrons are in
hydrogen-like orbitals
(s, p, d, f)
Molecules: electrons are in
molecular orbitals (HOMO,
LUMO, …)
From
http://education.jlab.org
(The Bohr model for nitrogen)
(The LUMO of benzene)
Molecular UV-Visible Spectroscopy
 Molecular UV-Visible spectroscopy is driven by electronic
absorption of UV-Vis radiation
 Molecular UV-Visible
spectroscopy can:
– Enable structural analysis
– Detect molecular chromophores
– Analyze light-absorbing properties
(e.g. for photochemistry)
 Basic UV-Vis spectrophotometers acquire data in the 190800 nm range and can be designed as “flow” systems.
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Molecular UV-Vis Spectroscopy: Terminology
 UV-Vis Terminology
– Chromophore: a UV-Visible absorbing functional group
– Bathochromic shift (red shift): to longer wavelengths
– Auxochrome: a substituent on a chromophore that
causes a red shift
– Hypsochromic shift (blue shift): to shorter wavelengths
– Hyperchromic shift: to greater absorbance
– Hypochromic shift: to lesser absorbance
Molecular UV-Vis Spectroscopy: Transitions
 Major classes of electron transitions
– HOMO: highest occupied molecular orbital
– LUMO: lowest unoccupied molecular orbital
– Types of electron transitions:
(1) ,  and n electrons (mostly organics)
(2) d and f electrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons
Molecular UV-Vis Spectroscopy: Theory
 Molecular energy levels and absorbance wavelength:
  * and   * transitions: high-energy, accessible in vacuum
UV (max <150 nm). Not usually observed in molecular UV-Vis.
n  * and   * transitions: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.
n  * and   * transitions: most common transitions observed in
organic molecular UV-Vis, observed in compounds with lone pairs
and multiple bonds with max = 200-600 nm.
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Molecular UV-Vis Spectroscopy and Transition
Metal and Lanthanide/Actinide Complexes
 d/f orbitals
– UV-Vis spectra of lanthanides/actinides are particularly sharp, due
to screening of the 4f and 5f orbitals by lower shells.
– Can measure ligand field strength, and transitions between dorbitals made non-equivalent by the formation of a complex
 Charge transfer (CT) – occurs when electron-donor and
electron-acceptor properties are in the same complex –
electron transfer occurs as an “excitation step”
– MLCT (metal-to-ligand charge transfer)
– LMCT (ligand-to-metal charge transfer)
– Ex: tri(bipyridyl)iron(II), which is red – an electron is exicted from
the d-orbital of the metal into a * orbital on the ligand
Molecular UV-Vis Spectroscopy: Absorption
 max is the wavelength(s) of maximum absorption (i.e. the

peak position)
The strength of a UV-Visible absorption is given by the
molar absorption coefficient ():
 = 8.7 x 1019 P a
where P is the transition probability (0 to 1) – governed by selection
rules and orbital overlap,
and a is the chromophore area in cm2
 Molar absorption coefficient () then gives A via the BeerLambert Law:
A = bc
Molecular UV-Vis Spectroscopy: Quantum Theory
 UV-Visible spectra and the states involved in electronic transitions

can be calculated with theories ranging from Huckel to ab initio/DFT.
Example:   * transitions responsible for ethylene UV absorption
at ~170 nm calculated with ZINDO semi-empirical excited-states
methods (Gaussian 03W):
HOMO u bonding molecular orbital
LUMO g antibonding molecular orbital
Molecular UV-Visible Spectrophotometers
 The traditional UVVis design: doublebeam grating
systems
 Sources:
 Almost

universal
continuum UVVis source is
the 2H lamp.
Tungsten lamps
used for longer
(visible)
wavelengths.
Hamamatsu
L2D2 lamps
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Molecular UV-Visible Spectrophotometers
 Diode array detectors can acquire all UV-Visible
wavelengths at once.
 Advantages:
– Sensitivity
(multiplex)
– Speed
 Disadvantages:
– Resolution
Figure from Skoog, et al., Chapter 13
Interpretation of Molecular UV-Visible Spectra
 UV-Visible spectra can be
interpreted to help determine
molecular structure, but this
is presently confined to the
analysis of electron behavior
in known compounds.
 Information from other
techniques (NMR, MS, IR) is
usually far more useful for
structural analysis
 However, UV-Vis evidence
should not be ignored!
Figure from Skoog, et al., Chapter 14
Calculation of Molar Absorption Coefficient
 The molar absorption coefficient () for each absorbance
in a UV spectrum is calculated as follows:
– , Molar Abs Coeff (AU mol-1 cm-1) = A x mwt / mass x pathlength
 Solvent “cutoffs” for UV-visible work:
Solvent
UV Cutoff (nm)
Acetonitrile (UV grade)
190
Acetone
330
Dimethylsulfoxide
268
Chloroform (1% ethanol)
245
Heptane
200
Hexane (UV grade)
195
Methanol
205
2-Propanol
205
Tetrahydrofuran (UV grade)
212
Water
190
Burdick and Jackson High Purity Solvent Guide, 1990
Interpretation of UV-Visible Spectra
 Although UV-Visible spectra are no longer frequently used
for structural analysis, it is helpful to be aware of welldeveloped interpretive rules.
 Examples:
– Woodward-Fieser rules for max dienes and polyenes
– Extended Woodward rules for unsaturated ketones
– Substituted benzenes (max base value = 203.5 nm)
X
Substituent (X)
Increment (nm)
-CH3
3.0
-Cl
6.0
-OH
7.0
-NH2
26.5
-CHO
46.0
-NO2
65.0
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
Interpretation of UV-Visible Spectra
 Other examples:
– The conjugation of a lone pair on a
enamine shifts the max from 190 nm
(isolated alkene) to 230 nm. The
nitrogen has an auxochromic effect.
H3C
H2N
CH2
vs.
~230 nm
HC
CH2
~180 nm
 Why does increasing conjugation cause bathochromic shifts
(to longer wavelengths)?
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Interpretation of UV-Visible Spectra
 Typical transition
metal complexes
show detailed
spectra, unlike
organic molecules
 Lanthanide
complexes show
sharp lines caused
by “screening” of
the f electrons by
other orbitals
See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14
Quantitative UV-Visible Spectroscopy
 UV-visible spectra can be used for direct quantitative
analysis with appropriate calibration
Ezetimibe calibration plot
Absorbace at 231 nm
1.2
y = 36.891x + 0.0814
R² = 0.9926
1
0.8
0.6
0.4
0.2
0
0
0.01
0.02
Concentration (M)
0.03
Color Analysis with Visible Spectra
 The visible region of a UV-Visible spectrum can be
decomposed into a color analysis (typically three numbers)
by simple calculations
– Involves multiplying the visible portion of the spectrum by color
functions and then taking the total area of the spectrum as a
single number
– Tristimulus values, which mimic the eye, are generally used and
then other values are determined from these algebraically
http://www.zeiss.de/c12567bb00549f37/ContentsFrame/80bd2fe43b50aa3ec125782c00597389
Diffuse Reflectance UV-Visible Spectroscopy
of Solids
 Solid powders can be studied using a diffuse reflectance
(DR) accessory either neat or diluted in a non-absorbing
powder
Diffuse Reflectance UV-Visible Spectroscopy
of Solids
 Typical diffuse reflectance spectrum of cyanocobalamin
(vitamin B12), diluted to 5% w/w in MgO
100
%Reflectance
80
60
40
20
0
250 300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Prediction of UV-Visible Spectra with Quantum
Calculations: Time-dependent DFT
 TDDFT:
Time-dependent density functional theory currently
provides accurate predictions of UV-visible spectra for
organic molecules
J. Mol. Struct. 2010, 984, 246–261, ttp://dx.doi.org/10.1016/j.molstruc.2010.09.036
Plane (or Linearly) Polarized Light
 If the electric vector of an EM wave points in the same
direction as that of the wave propagating through a
medium, the light is said to be linearly polarized
T0
K  log10
T90
Figure from Sears, et al., “University Physics”, 7th Ed., 1988
Polarimetry and Optical Rotation
 A polarimeter measures the
angle of rotation of linearly
polarized monochromatic light
as it passes through a sample
– Source: sodium arc lamp (589
nm), now commonly replaced
with a yellow LED
– Two polarizers before and after
the sample. One is fixed and
the other is rotated to find the
maximum light transmitted, and
the rotation is recorded.
– Result is a single number, e.g.
-10.02, the specific rotation
– What happens when we vary
the wavelength?
Optical Rotation and ORD
 The rotation of plane polarized light by molecules:
Eliel et al., “Stereochemistry of Organic Compounds”, p. 997.
R. P Feynman, et al., “The Feynman Lectures on Physics”, 1963, Addison-Wesley. p. 33-6
Optical Rotatory Dispersion (ORD)
 The measurement of specific rotation as a function of
wavelength, in the absence of absorption, is monotonic
(and governed by the Fresnel equation)
 In the vicinity of an absorption, one obtains “anomalous
dispersion”
UV-Visible Circular Dichroism
 UV-visible or electronic circular dichroism (ECD or just
CD) is the study of differential absorption of polarized UVVisible radiation by chiral molecules.
 CD measures the difference between LCPL and RCPL
 Beer’s law for CD:
A = bc
 Where  = (LPCL - RPCL)
 is the molar absorptivity (cm-1 M-1)
A is absorption
See Eliel, et al. Stereochemistry of Organic Compounds, pg. 1003.
Circularly-Polarized UV-Visible Radiation
 Circularly-polarized UV-visible radiation is made by mixing
two orthogonal electric field components 90 degrees out
of phase.
 In practice, a quartz crystal is subjected to mechanical
stress and (via the piezoelectric effect) causes circular
polarization of the light
Animation from http://www.bip.bham.ac.uk/osmart/bcm201_cd/cd_movie/index.html
UV-Visible Circular Dichroism
 A typical UV-Visible CD spectrometer, the Jasco J-715
Electronic Circular Dichroism
 CD spectra of (1S)-(+)-10-camphorsulfonic acid and (1R)(+)-10-camphorsulfonic acid (ammonium salts) in H2O
200
100
CD[mdeg]
0
-100
-200
1000
800
HT[V]
600
400
200
190
250
300
Wavelength [nm]
350
TDDFT Calculations
 TDDFT calculations have largely replaced empirical rules.
 Example: (1R)-(+)-10-camphorsulfonic acid (ammonium
salts) and its isomer calculated without solvation:
TDDFT ECD B3LYP/6-311+G(2d,p) 50-50
3
1R-10-camphorsulfonic acid
ammonium salt
2
Rvel (10 -40 esu2 cm 2 )
1S-10-camphorsulfonic acid
ammonium salt
1
0
-1
-2
-3
200
220
240
260
280
300
320
340
Excitation wavelength (nm)
360
380
400
420
440
Electronic Circular Dichroism
 Variable temperatuer CD spectra of an orally-bioavailable
PTH mimetic peptide, showing conformational changes:
1
16
31
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-LeuAsn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-AspVal-(NH2)
Anal. Chem. 2012, 84, 4357-4372, http://dx.doi.org/10.1021/ac203478r
Electronic Circular Dichroism
 ECD has extensive applications to structural analysis in
proteins, antibodies, and other biopolymers
N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.
Electronic Circular Dichroism
 Different protein conformations give rise to different spectra
 CD spectra are numerically fitted to extract conformational
population
N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.
Hyphenated Circular Dichroism Experiments
Example: Related
atropoisomeric
compounds
studied in
stopped-flow LCCD experiments
T. J. Edkins and D. R. Bobbitt, Anal. Chem., 2001, 73, 488A-496A
G. Bringmann, et al., Anal. Chem., 1999, 71, 2678-2686.
The Cotton Effect
 The Cotton effect:
– An extrema in the ECD spectrum
– Or, a zero-crossing in the ORD spectrum
Other Notes on Electronic Circular Dichroism
 Background signals – UV absorbance that does not
depend on the polarization constitutes the background
(Dynamic Reserve).
 DR = A/A = / = /(LPCL - RPCL)
 is the molar absorptivity (cm-1 M-1)
A is absorption
 DR values of 2 x104 are possible
 Electronic background suppression is almost always
used instead of optical background suppression
(technical design issues)
Elliptically Polarized Light
 Combining left and right circularly polarized waves of
unequal amplitudes = elliptically polarized light
 Basis of ellipsometry – a surface analysis method used to
study:
–
–
–
–
–
Layer/film thickness
Optical constants (refractive index and extinction coefficient)
Surface roughness
Composition
Optical anisotropy
Further Reading
Optional:
J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Edition, 2005, Marcel
Dekker, Chapters 5 and 6.
D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis, 6th Edition,
2006, Brooks-Cole, Chapters 13 and 14.
D. H. Williams and I. Fleming, “Spectroscopic Methods in Organic Chemistry”, McGraw-Hill
(1966).
D. A. Lightner and J. E. Gurst, “Organic Conformational Analysis and Stereochemistry from
Circular Dichroism Spectroscopy,” Wiley-VCH, 2000.